The epidermal growth factor receptor (EGFR) is often described as a `prototypic' receptor tyrosine kinase (RTK), and its activation is typically considered to occur through straightforward growth factor-induced receptor dimerization. With such a simple model, however, it is difficult to explain how different activating ligands such as TGFa, betacellulin, HB-EGF, epiregulin, epigen, and amphiregulin can elicit distinct cellular signals through the same receptor - possibly through different patterns of autophosphorylation or through trafficking differences. Moreover, a simple dimerization model cannot explain how mutationally activated forms of EGFR in lung cancer (with kinase mutations) and glioblastoma (with extracellular missense mutations or deletions as in EGFRvIII) would have distinct inhibitor sensitivities, as has been reported. These observations and many others suggest that EGFR is a complex allosterically regulated receptor, which we know binds ligands with negative cooperativity. We are therefore focused on understanding allosteric regulation of this receptor in detail, with a view to understanding EGFR signaling specificity and identifying which oncogenic mutations promote sensitivity to which classes of EGFR inhibitors. These studies may also suggest new strategies for targeted therapeutics. To gain insight into the communication of allosteric changes across the membrane, we propose structural and biochemical studies of EGFR activated by its different ligands - or by oncogenic mutations - using a variety of techniques. Importantly, we will also analyze the receptor's conformational dynamics using hydrogen/deuterium exchange mass spectrometry (HX-MS). HX-MS studies will be performed for isolated extracellular regions of wild-type and mutated receptors and for intact EGFR activated by ligands or mutations - to provide insight into conformational fluctuations in the extracellular domain, kinase domain, and linking regions. We will determine how ligand binding or oncogenic mutation alters the conformation, dynamics, and extent of activation of the intact EGFR, and how these changes are propagated across the membrane. Our preliminary data also suggest that HX-MS analyses will provide valuable insight into the role of the disordered carboxy-terminal tail, and how it might contribute to signaling specificity. Our overall goal is t bridge the current gulf between the structural information about EGFR family members, our understanding of their transmembrane signaling properties in cells, and mechanisms of activation (and inhibition) in cancer. Our Specific Aims address two main questions: 1 how do activated complexes of EGFR induced by its seven different ligands differ in structure, activity, and dynamics? 2 How do oncogenic forms of EGFR found in glioblastoma compare in structure, activity, and dynamics to the ligand-activated receptor - and how do differences lead to altered kinase inhibitor selectivity?